1. Field of the Invention
This invention relates to a hybridoma cell line and monoclonal antibody produced therefrom which may be used to detect the .beta.-adrenergic agonist, ractopamine and its metabolites, particularly in animal tissues, excreta, feeds, and in human excretions.
2. Description of the Prior Art
Ractopamine is a phenethanolamine leanness-enhancing agent recently approved as a feed additive for swine by the United States Food and Drug Administration Center for Veterinary Medicine (Federal Register, 65, 4111-4112, 2000; Muirhead, Feedstuffs, 72,1, 2000). Hogs administered dietary ractopamine exhibit increased growth rates, feed efficiencies, and greater yields of boneless, closely trimmed retail cuts (Anderson et al., Fat and cholesterol reduced foods: technologies and strategies, 43-73, 1990; Stites et al., J. Anim. Sci., 69, 3094-3101, 1991; Watkins et al., J. Anim. Sci., 68, 3588-3595, 1990) relative to untreated control animals. The positive influence of ractopamine hydrochloride on these economically important traits should make the product attractive to swine growers, and perhaps to producers of livestock species for which ractopamine is not approved.
Phenethanolamine .beta.-agonists have a history of being used for off-label purposes by livestock producers (Kuiper et al., J. Anim. Sci., 76, 195-207, 1998; Mitchell et al., J. Anim. Sci., 76, 208-211, 1998) hoping to improve the economics of livestock production; additionally, they have been used extensively by body-builders hoping to modify their phenotypic characteristics (Prather et al., 27, 1118-1121, 1995; Hausmann et al., Int. J. Legal Med., 111:261-264, 1998; Ayotte et al., J. Toxicol.-Toxin Rev., 18, 113-123, 1999). The most commonly abused .beta.-agonist is clenbuterol hydrochloride, a highly potent phenethanolamine (Smith, J. Anim. Sci., 76, 173-194, 1998), that has not been approved for leanness-enhancing effects in livestock or in humans by any regulatory body worldwide.
The presence of drug residues in animal tissues is a concern for food safety, especially when the compound has been used illegally or in a manner proscribed by regulatory officials (off-label use). In an effort to combat the illicit use of .beta.-agonist compounds, regulatory organizations worldwide test animal tissues or excreta for the presence of illicit drugs (Elliott et al., Vet. Quart., 18, 41-44, 1996; Kuiper et al., J. Anim. Sci., 76, 195-207, 1998; Mitchell et al., J. Anim. Sci., 76, 208-211, 1998). For regulatory purposes, both screening and confirmatory assays are used to detect illegal residues. Immunoassays are convenient screening tools used to detect the presence of an analyte in various matrices, and have wide application for determination of the presence of environmental toxins (Sanborn et al., J. Agric. Food. Chem., 46, 2407-2416, 1998), herbicides (Clegg et al., J. Agric. Food. Chem., 47, 5031-5037, 1999), insecticides/pesticides (Wang et al., J. Agric. Food Chem., 47, 3416-3424, 1999; Abad et al., J. Agric. Food. Chem., 47, 2475-2485, 1999), and pharmaceuticals (Stanker et al., Food Agric. Immuno., 10, 121-131, 1998; Brandon et al., J. Agric. Food Chem., 46, 3653-3656, 1998). A successful screening assay should be quick, reliable, and relatively inexpensive. Positive samples from screening assays may then be assayed by more costly and complex instrumental methods such as GC-MS or LC-MS that unequivocally identify the analyte in the sample (eliminating false-positives). However, for screening, immunoassays provide the advantages of high throughput, portability, and sensitivity (detection limits in the ppb range). High sensitivities of immunoassays are particularly desirable for off-label drug monitoring because it may be desirable to detect the analyte even after extended withdrawal periods. Beta-adrenergic agonists immunoassays have been developed for clenbuterol (Yamamoto et al., J. Immunoassay, 3: 155-171, 1982), albuterol (salbutamol; Adam et al., J. Immunoassay, 11, 329-345, 1990), fenoterol (Haasnoot et al., Analyst, 119, 2675-2680, 1994), as well as ractopamine (Haasnoot et al., Analyst, 119, 2675-2680, 1994; Elliott et al., Analyst, 123, 1103-1107, 1998; Shelver et al., J. Immunoassay, 21, 1-23, 2000). Currently available immunoassay kits cross-react poorly with ractopamine (Wicker et al., Analyst, 120, 2879-2881, 1995), and the immunoassay previously reported for ractopamine was generated from polyclonal antibodies. Development of an immunoassay based on a monoclonal antibody is advantageous, relative to a polyclonal based immunoassay because a continuous supply of a homogeneous antibody can be assured, eliminating the batch-to-batch differences commonly encountered with polyclonal antibodies.